WRKY6 transcription factor restricts arsenate uptake and transposon activation in Arabidopsis.

Stress constantly challenges plant adaptation to the environment. Of all stress types, arsenic was a major threat during the early evolution of plants. The most prevalent chemical form of arsenic is arsenate, whose similarity to phosphate renders it easily incorporated into cells via the phosphate transporters. Here, we found that arsenate stress provokes a notable transposon burst in plants, in coordination with arsenate/phosphate transporter repression, which immediately restricts arsenate uptake. This repression was accompanied by delocalization of the phosphate transporter from the plasma membrane. When arsenate was removed, the system rapidly restored transcriptional expression and membrane localization of the transporter. We identify WRKY6 as an arsenate-responsive transcription factor that mediates arsenate/phosphate transporter gene expression and restricts arsenate-induced transposon activation. Plants therefore have a dual WRKY-dependent signaling mechanism that modulates arsenate uptake and transposon expression, providing a coordinated strategy for arsenate tolerance and transposon gene silencing.

The potassium transporters HAK2 and HAK3 localize to endomembranes in Physcomitrella patens. HAK2 is required in some stress conditions.

The function of HAK transporters in high-affinity K+ uptake in plants is well established; this study aims to demonstrate that some transporters of the same family play important roles in endomembranes. The PpHAK2-PpHAK4 genes of Physcomitrella patens encode three transporters of high sequence similarity. Quantitative PCR showed that PpHAK2 and PpHAK3 transcripts are expressed at approximately the same level as the PpACT5 gene, while the expression of PpHAK4 seems to be restricted to specific conditions that have not been determined. KHA1 is an endomembrane K+/H+ antiporter of Saccharomyces cerevisiae, and the expression of the PpHAK2 cDNA, but not that of PpHAK3, suppressed the defect of a kha1 mutant. Transient expression of the PpHAK2-green fluorescent protein (GFP) and PpHAK3-GFP fusion proteins in P. patens protoplasts localized to the endoplasmic reticulum and Golgi complex, respectively. To determine the function of PpHAK2 and PpHAK3 in planta, we constructed ΔPphak2 and ΔPphak2 ΔPphak3 plants. ΔPphak2 plants were normal under all of the conditions tested except under K+ starvation or at acidic pH in the presence of acetic acid, whereupon they die. The defect observed under K+ starvation was suppressed by the presence of Na+. We propose that PpHAK2 may encode either a K(+)-H(+) symporter or a K+/H+ antiporter that mediates the transfer of H+ from the endoplasmic reticulum lumen to the cytosol. PpHAK2 may be a model of the second function of HAK transporters in plant cells. The disruption of the PpHAK3 gene in ΔPphak2 plants showed no effect.

Potassium and sodium uptake systems in fungi. The transporter diversity of Magnaporthe oryzae.

In this study, we report an inventory of the K(+) uptake systems in 62 fungal species for which the complete genome sequences are available. This inventory reveals that three types of K(+) uptake systems, TRK and HAK transporters and ACU ATPases, are widely present in several combinations across fungal species. PAT ATPases are less frequently present and are exceptional in Ascomycota. The genome of Magnaporthe oryzae contains four TRK, one HAK, and two ACU genes. The study of the expression of these genes at high K(+), K(+) starvation, and in infected rice leaves revealed that the expression of four genes, ACU1, ACU2, HAK1, and TRK1 is much lower than that of TRK2, TRK3, and TRK4, except under K(+) starvation. The two ACU ATPases were cloned and functionally identified as high-affinity K(+) or Na(+) uptake systems. These two ATPases endow Saccharomyces cerevisiae with the capacity to grow for several generations in low Na(+) concentrations when K(+) was absent, which produces a dramatic increase of cellular Na(+)/K(+) ratio.

The SOS1 transporter of Physcomitrella patens mediates sodium efflux in planta.

• SOS1 is an Na(+)/H(+) antiporter that plays a central role in Na(+) tolerance in land plants. SOS1 mediation of Na(+) efflux has been studied in plasma-membrane vesicles and deduced from the SOS1 suppression of the Na(+) sensitivity of yeast mutants defective in Na(+) -efflux. However, SOS1-mediated Na(+) efflux has not been characterized in either plant or yeast cells. Here, we use Physcomitrella patens to investigate the function of SOS1 in planta. • In P. patens, a nonvascular plant in which the study of ion cellular fluxes is technically simple, the existence of two SOS1 genes suggests that the Na(+) efflux remaining after the deletion of the ENA1 ATPase is mediated by a SOS1 system. Therefore, we cloned the P. patens SOS1 and SOS1B genes (PpSOS1 and PpSOS1B, respectively) and complementary DNAs, and constructed the PpΔsos1 and PpΔena1/PpΔsos1 deletion lines by gene targeting. • Comparison of wild-type, and PpΔsos1 and PpΔena1/PpΔsos1 mutant lines revealed that PpSOS1 is crucial for Na(+) efflux and that the PpΔsos1 line, and especially the PpΔena1/PpΔsos1 lines, showed excessive Na(+) accumulation and Na(+)-triggered cell death. The PpΔsos1 and PpΔena1/PpΔsos1 lines showed impaired high-affinity K(+) uptake. • Our data support the hypothesis that PpSOS1 mediates cellular Na(+) efflux and that PpSOS1 enhances K(+) uptake by an indirect effect.

Role of ENA ATPase in Na(+) efflux at high pH in bryophytes.

Potassium or Na(+) efflux ATPases, ENA ATPases, are present in all fungi and play a central role in Na(+) efflux and Na(+) tolerance. Flowering plants lack ENA ATPases but two ENA ATPases have been identified in the moss Physcomitrella patens, PpENA1 and PpENA2. PpENA1 mediates Na(+) efflux in Saccharomyces cerevisiae. To propose a general function of ENA ATPases in bryophytes it was necessary to demonstrate that these ATPases mediate Na(+) efflux in planta and that they exist in more bryophytes than P. patens. For these demonstrations (1) we cloned a third ATPase from P. patens, PpENA3, and studied the expression pattern of the three PpENA genes; (2) we constructed and studied the single and double Deltappena1 and Deltappena2 mutants; and (3) we cloned two ENA ATPases from the liverwort Marchantia polymorpha, MpENA1 and MpENA2, and expressed them in S. cerevisiae. The results from the first two approaches revealed that the expression of ENA ATPases was greatly enhanced at high pH and that Na(+) efflux at high pH depended on PpENA1. The ENA1 ATPase of M. polymorpha suppressed the defective growth of a S. cerevisiae mutant at high K(+) or Na(+) concentrations, especially at high K(+).

Effects of polylinker uATGs on the function of grass HKT1 transporters expressed in yeast cells.

HvHKT1 mediates K(+) or Na(+) uniport in yeast cells if the expression promoter is joined directly to the HvHKT1 cDNA, and Na(+)-K(+) symport if a 59 nucleotide polylinker is inserted. Our results show that three ATG triplets in the polylinker decreased the synthesis of the transporter and that the lower amount of transporter caused the functional change. With the rice HKT1 cDNA, the 59 nt polylinker changed the mode of Na(+) uptake from K(+)-insensitive to K(+)-inhibitable. These two modes of Na(+) uptake also occurred in rice plants.